Electroporation

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.cite journal |author=Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH |title=Gene transfer into mouse lyoma cells by electroporation in high electric fields |journal=EMBO J. |volume=1 |issue=7 |pages=841–5 |year=1982 |pmid=6329708 |doi=]

Pores are formed when the voltage across a plasma membrane exceeds its dielectric strength. If the strength of the applied electrical field and/or duration of exposure to it are properly chosen, the pores formed by the electrical pulse reseal after a short period of time, during which extracellular compounds have a chance to enter into the cell. However, excessive exposure of live cells to electrical fields can cause "apoptosis" and/or "necrosis" - the processes that result in cell death.

In molecular biology, the process of electroporation is often used for the "transformation" of bacteria, yeast, and plant protoplasts. In addition to the lipid membranes, bacteria also have cell walls which are different from the lipid membranes and are made of peptidoglycan and its derivatives. However, the walls are naturally porous and only act as stiff shells that protect bacteria from severe environmental impacts. If bacteria and plasmids are mixed together, the plasmids can be transferred into the cell after electroporation. Several hundred volts across a distance of several millimeters are typically used in this process. Afterwards, the cells have to be handled carefully until they have had a chance to divide producing new cells that contain reproduced plasmids. This process is approximately ten times as effective as "chemical transformation".cite journal |author=Sugar IP, Neumann E |title=Stochastic model for electric field-induced membrane pores. Electroporation |journal=Biophys. Chem. |volume=19 |issue=3 |pages=211–25 |year=1984 |pmid=6722274| doi = 10.1016/0301-4622(84)87003-9 ]

This procedure is also highly efficient for the introduction of foreign genes in tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process of introducing foreign DNAs into eukaryotic cells is known as transfection.

The process

Electroporation is done with electroporators, appliances which create the electric current and send it through the cell solution (typically bacteria, but other cell types are sometimes used, as discussed above). The solution is pipetted into a glass or plastic cuvette which has two aluminum electrodes on its sides.

For instance, for bacterial electroporation, a suspension of around 50 microliters is usually used. Prior to electroporation it is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage is set on the electroporator (240 volts is often used) and the cuvette is inserted into the electroporator. Immediately after electroporation 1 milliliter of liquid medium is added to the bacteria (in the cuvette or in an eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more and then it is spread on an agar plate.

The success of the elecroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Impure solutions might cause a small explosion (known as arcing), in which case the bacteria are dead and the process needs to be redone. If this happens often, an additional precipitation may be performed prior to electroporation.

Medical Applications

A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within a narrow range while leaving neighboring cells unnaffected, and thus represents a promising new treatment for cancer, heart disease and other disease states that require removal of tissue. [cite web |url= http://www.berkeley.edu/news/media/releases/2007/02/12_IRE.shtml|title=New medical technique punches holes in cells, could treat tumors |accessdate=2007-12-13 |author=Sarah Yang|date=2007-02-12]

References